Technologies in Clinical Liquid Chromatography

D-binding immunoaffinity solid phase is used in a batch mode to concentrate vitamin D analogs prior to reverse-phase LC separation and mass spectrometric quantification [16]. Alternatively, an immunoaffinity column can be used in the main separation step of analysis, as has been reported for analysis of transferrin isoforms [17], An important consideration in the use of immunoaffinity columns in the clinical laboratory is the increased cost of the method compared to more common solid phases and the fact that the life time of an immunoaffinity column is shorter than a standard reversed- or normal-phase column. 

UV—visible spectrophotometry is a convenient detection methodology for liquid chromatography in the clinical laboratory because of the low cost and reliability of the detector and because many small molecules of interest in the clinical laboratory absorb ultraviolet light. Absorbance is one of the least sensitive detection modalities, however, so some low-abimdance drug and hormone analytes can be difficult to detect. 

Several small molecules of clinical interest contain highly conjugated systems and fimctional groups that are amenable to redox chemistry. These molecules, such as the catecholamines [19], are separated by reversed phase chromatography and then participate in a redox reaction 

Additional developments in mass analysis technology, including ion traps and time-of-flight (TOP) mass analyzers, have allowed measurements of molecular mass with sufficient resolution to identify compounds by their exact mass. While these technologies have not penetrated the clinical laboratory significantly at the time of this writing, they are likely to play an important role in the next generation of clinical assays. 

Other therapeutic drug classes currently quantified by LC—MS in clinical laboratories include antiepileptic medications (levetiracetam [23]), immunosuppressive/antivirals (leflunomide [24]), and synthetic steroids (prednisolone, methylprednisolone, dexamethasone, and others [25]). 

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Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

During the last decade, the use of liquid chromatography-mass spectrometry (LC-MS) technology has been increasing in clinical and forensic laboratories. This expansion is in part because of the gradual introduction of relatively low-cost LC-MS instruments and the availability of robust atmospheric pressure ionization (API) sources. The combination of liquid chromatographic separation and mass spectrometric detection provides the laboratory an efficient... 

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